Though often considered to be a disease of the past, tuberculosis (TB) is still a leading killer of people worldwide. At the end of 2008, approximately 2 billion people one-third of the world's population is infected with Mycobacterium tuberculosis (MTb), the etiologic agent of TB. The World Health Organization estimates that 2 million people worldwide die of TB each year. TB is now the second most common cause of death from infectious disease in the world after human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). In the mid-1980s, a resurgence of outbreaks in the United States brought renewed attention to TB. Main reasons for increase in TB cases are due to higher vulnerability of people infected with HIV/AIDS and development of drug-resistant strains of MTb, which causes TB in human. In the latest 2010 April WHO report results show that one in every 4 TB patients diagnosed with MDR-TB in Russia and about 50% of MDR-TB burden is in China and India. Worldwide there are 440,000 MDR-TB cases and 150,000 deaths occurred in 2008. About 5.4% of MDR-TB cases have XDR-TB according to the report in March 2010.It is impractical to eliminate TB without novel, and more effective drugs, diagnostics and vaccines. The world immediately needs simpler and faster curative drugs;safer and effective drugs that can treat all forms of TB especially in immuno- compromised people with HIV/TB co-infections. As an alternative to antibiotic therapy, especially for systemic lung infections by M. tuberculosis, we are proposing to develop an enzyme therapy that can be used with various formulation conditions and delivery routes. The enzyme therapy may be as effective as antibiotic therapy because the enzyme is highly specific, and potent against the targeted bacteria. However, delivering these enzymes to the site of the infection remains a challenge. Most pharmaceutical proteins are susceptible to degradation at the site of administration, whether administered intravenously, subcutaneously, orally, topically or by any other route. We believe that a safe and effective delivery system for this application may be through the use of protein crystals, which provides highly concentrated, stable, and pure products. In addition, technologies are also needed to deliver endolysin to intracellular compartment since M. tuberculosis resides in macrophages. Cell penetrating peptides (CPP), either engineered in-frame or adsorbed/bound non-covalently to protein substrates, have been used to deliver a wide variety of molecules into a number of cell types. In this proposal we are proposing to develop a crystalline formulated recombinant endolysin therapy with cell-penetrating peptides (CPP) that can be used with various formulation conditions and delivery routes. The opportunities for CPP conjugated to endolysin are multiple since it may be able to penetrate the macrophages thereby able to kill M. tuberculosis in HIV/TB co-infected patients and may extend their life span with minimal discomfort. In Phase I, we intend to study the feasibility of using endolysins, both soluble and crystalline formulations with or without cell penetrating peptides, as a systemic delivery system in an in vivo animal model for M. tuberculosis infection. If successful, this approach will lead to the introduction of novel, efficient enzyme therapy for the treatment of M. tuberculosis infections. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) is one of the most devastating global health problems of our time, causing approximately 2 million deaths a year. As a preventive measure, currently BCG vaccine is used to avoid serious complications of TB and to reduce the rate of pediatric TB but It has little or no effectiveness for pulmonary TB in adults. We are proposing a novel crystalline endolysin that is pure, safe, effective even in immunocompromised patients, and unlike vaccines which needs immune system to generate the antibodies the endolysin can act directly on the Mycobacterium tuberculosis organism and thus prevent and treat tuberculosis in HIV/TB co-infected patients.